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Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A
P Yaciuk1, J K Choi, D Shalloway
1Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Abstract:
The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.
Insights
Phosphorylation at Ser-12 and Ser-17 sites of pp60c-src does not significantly impact its transforming activity in NIH 3T3 cells. However, Ser-17 phosphorylation might play a subtle regulatory role in c-src proto-oncogene function.
Area of Science:
- Molecular Biology
- Cellular Biology
- Oncology
Background:
- The c-src proto-oncogene product, pp60c-src, is a key regulator of cellular processes.
- pp60c-src is phosphorylated by protein kinase A at Ser-17 and protein kinase C at Ser-12.
- Understanding these phosphorylation events is crucial for deciphering c-src's role in cell transformation.
Purpose of the Study:
- To investigate the functional impact of mutations at Ser-12 and Ser-17 on pp60c-src and its enhanced mutant pp60c-src(F527).
- To determine if altering these phosphorylation sites affects the kinase activity and transforming potential of pp60c-src.
- To explore potential cross-regulation between Ser-12 and Ser-17 phosphorylation.
Main Methods:
- Site-directed mutagenesis was used to create Ser12Ala, Ser17Ala, and combined mutations in pp60c-src and pp60c-src(F527) expression plasmids.
- Mutant plasmids were transfected into NIH 3T3 cells for analysis.
- Tryptic phosphopeptide analysis was performed to confirm phosphorylation sites and assess effects of mutations.
- Kinase activity assays and focus formation assays were conducted to evaluate transforming potential.
Main Results:
- Mutations at Ser-12 and Ser-17 did not affect phosphorylation at other key sites (Tyr-416, Tyr-527, Ser-48) or mitosis-specific phosphorylations.
- Mutation of Ser-17 increased phosphorylation at Ser-12, suggesting a potential regulatory interaction.
- Neither pp60c-src nor pp60c-src(F527) transforming activities (foci formation, transformed morphology, anchorage-independent growth) were significantly altered by the serine mutations.
- Kinase activity remained largely unaffected by the mutations, with or without phorbol ester treatment.
Conclusions:
- Phosphorylation at Ser-12 and Ser-17 of pp60c-src has a limited impact on its transforming activity in NIH 3T3 cells.
- Ser-17 phosphorylation may exert a subtle regulatory influence on pp60c-src function.
- The study highlights the relative insensitivity of pp60c-src transforming potential to modifications at these specific serine residues.
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